Internal fertilization is a complex biological process that requires interactions between proteins provided by both males and females. In addition to proteins found on the sperm and the egg, additional accessory proteins from seminal fluid and the female reproductive tract are required for successful reproduction. Because the functions of these proteins directly impact reproductive success, reproductive proteins experience strong evolutionary selective pressures, and interacting proteins are predicted to coevolve with one another. However, while reproductive proteins from both sexes have been readily identified, how these proteins interact is a relatively open question. This proposal seeks to investigate these issues in the genetically tractable Drosophila melanogaster (fruit fly) model system, which bears important parallels to human and mammalian reproductive systems. The seminal fluid proteins (Sfps) found in Drosophila males belong to the same functional classes as those identified in mammalian taxa, and many Drosophila male and female reproductive proteins show the same evolutionary dynamics as those from other systems. Thus, the methods used to study the functions of Drosophila proteins and the types of interactions involving these proteins may generate useful techniques and hypotheses for studying reproductive proteins and infertility in other systems. In order to address reproductive protein function, interaction and coevolution, this proposal will investigate as a case study a network of Sfps that together regulate mated females' egg production, sperm storage and mating behavior. Particular focus will be paid to proteins that interact molecularly with a well-characterized Sfp, the sex peptide (SP), to mediate its association with and release from sperm. The research will use two complementary methods to identify other Sfps, sperm proteins, and female proteins that interact with SP and other components of this network. The first method is a novel phylogenetic approach that uses correlated evolutionary rates to predict protein-protein interactions. The second involves biochemical pull-down assays to identify interacting sperm and female proteins that act at critical steps in the pathway. Candidate interacting proteins identified from either method will be tested molecularly and genetically to determine how they interact with and regulate SP and, thereby, mediate female post-mating responses. These experiments will offer important insights into basic reproductive biology and the study of protein-protein interactions, while providing critical and novel training to the applicant in areas of genetics, biochemistry and evolutionary biology.